Chronic graft-versus-host disease (GVHD) is the most common cause of poor long-term outcomes after allogeneic bone marrow transplantation (BMT), but the pathophysiology of chronic GVHD still remains poorly understood. We tested the hypothesis that the impaired thymic negative selection of the recipients will permit the emergence of pathogenic T cells that cause chronic GVHD. Lethally irradiated C3H/HeN (H-2k) recipients were reconstituted with T-cell-depleted bone marrow cells from major histocompatibility complex [MHC] class II-deficient (H2-Ab1-/-) B6 (H-2b) mice. These mice developed diseases that showed all of the clinical and histopathological features of human chronic GVHD. Thymectomy prevented chronic GVHD, thus confirming the causal association of the thymus. CD4+ T cells isolated from chronic GVHD mice were primarily donor reactive, and adoptive transfer of CD4+ T cells generated in these mice caused chronic GVHD in C3H/HeN mice in the presence of B6-derived antigen-presenting cells. Our results demonstrate for the first time that T cells that escape from negative thymic selection could cause chronic GVHD after allogeneic BMT. These results also suggest that self-reactivity of donor T cells plays a role in this chronic GVHD, and improvement in the thymic function may have a potential to decrease chronic GVHD.